The KINOMEscan assay platform is based on a competition binding assay that is run for a compound of interest against each of a panel of 442 kinases. The assay has three components: a kinase-tagged phage, a test compound, and an immobilized ligand that the compound competes with to displace the kinase. The amount of kinase bound to the immobilized ligand is determined using quantitative PCR of the DNA tag. Kd values (nM) reported for each compound were determined using 11 serial threefold dilutions of test compound and a DMSO control. A null result means no inhibition of kinase binding to the ligand in the presence of the compound and low Kd means strong inhibition.

Assay Protocol:

Competition binding assays were developed, validated and performed as described in Fabian, et al. 2005, Nat Biotechnol.. Protocol Summary from Davis, et al. 2011: Kinases were produced either as fusions to T7 phage3, or were expressed as fusions to NF-κB in HEK-293 cells and subsequently tagged with DNA for PCR detection18. In general, full-length constructs were used for small, single-domain kinases, and catalytic domain constructs including appropriate flanking sequences were used for multidomain kinases. Briefly, for the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands to generate affinity resins. The liganded beads were blocked to reduce nonspecific binding and washed to remove unbound ligand. Binding reactions were assembled by combining kinase, liganded affinity beads and test compounds prepared as 100× stocks in DMSO. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in 384-well plates, whereas Kd determinations were performed in 96-well plates. Assay plates were incubated at 25 °C with shaking for 1 h, and the affinity beads were washed extensively to remove unbound protein. Bound kinase was eluted in the presence of nonbiotinylated affinity ligands for 30 min at 25 °C with shaking. The kinase concentration in the eluates was measured by quantitative PCR. Kds were determined using 11 serial threefold dilutions of test compound and a DMSO control.